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Problems with Ecoli glycerol stock


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#1 krisw

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Posted 05 November 2012 - 07:40 AM

Hi everyone,

I've been at this job for 4 years and have recently started having problems with my glycerol stocks. We freeze down 15% glycerol Ecoli stocks at -80. The weird part is that when I go to streak that stock on a plate, the cells grow just fine. Then, if I pick an individual colony for liquid culture, it does not grow. This has happened recently with many different samples and so I dont believe it was just a one time mistake (for example, if I just happened to pick a bad colony).
If I take my actual purified DNA, re-transform and then pick a colony and grow that in liquid cultrue, they grow fine. So, I have no reason to suspect that my media or antibiotics are bad. I am thinking that something is wrong with the actual glycerol stocks. But what would cause the cells to grow on a plate but not in liquid culture? Could old glycerol all of a sudden cause such problem (ours is about 6 years old)?
Has anyone ever had this happen? I plan to buy a new glycerol batch and do some testing, but I want yo get your thoughts to see if I'm on the right track here.

Thanks, Kris

#2 bob1

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Posted 05 November 2012 - 12:49 PM

Have your glycerols been thawed by accident?

#3 krisw

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Posted 06 November 2012 - 01:32 PM

I have no reason to believe our stocks have been thawed. Our freezers have alarms on them.

#4 ascacioc

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Posted 06 November 2012 - 04:02 PM

yeah... but you don't know who took them out and in after 1-2 hours in the weekend without saying anything (I have seen similar things happening). I would inoculate in a liquid culture directly from the stock and see what happens. On the other hand, I would never trust that culture if I do not see single bacterial colony on the plate. On the other hand, if you still have the DNA, what would stop you from getting a new transformation? It might be a bit too much work, but better do it again than forever wonder whether what you are working with is the real thing and about where the problem is. Just my two obsessive compulsive-paranoid cents...

Andreea

#5 pito

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Posted 08 November 2012 - 10:00 AM

So, if understanding correctely:

you pick some of the stock, plate it out on solid media: grows
you then pick a colony from the plate: no growth?

Have you tried to pick a colony from the plate to plate this out one more?
(thus 2 plates before inoculation a liquid culture)

+ you do wait long enough for it to grow? Because in liquid it can take a while, its just 1 colony in one gigantic liquid sample...

+ you do use the same technique for picking the colony for the liquid and plate ?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#6 krisw

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Posted 19 November 2012 - 09:38 AM

Thanks for all the help so far.
Ascacioc: I have had to resort to doing transformations. This is the only way I can get around this problem, but, of course, it's much more time and money to have to do so.
Pito: Yes, you got that right. Colonies grow on plates, but not liquid culture. I did a transformation and then grew that in liquid culture side by side with the glycerol stock colony. The transformation one growns just fine and glycerol does not. So, growing time does not seem to be a problem. Technique is exactly the same. I have not tried to do 2 plates before the liquid culture, something I may need to do.

I also just tested a new batch of glycerol and got the same results. I was fairly hopeful that that was the problem, but the new glycerol did not help. I really can't think of why the cells would grow on the plate but not in culture. And why it just started happening all of a sudden. (I grew liquid cultures from glycerol back in July, then the next time in September it failed to work).

Any more ideas?

#7 pito

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Posted 19 November 2012 - 11:10 AM

Thanks for all the help so far.
Ascacioc: I have had to resort to doing transformations. This is the only way I can get around this problem, but, of course, it's much more time and money to have to do so.
Pito: Yes, you got that right. Colonies grow on plates, but not liquid culture. I did a transformation and then grew that in liquid culture side by side with the glycerol stock colony. The transformation one growns just fine and glycerol does not. So, growing time does not seem to be a problem. Technique is exactly the same. I have not tried to do 2 plates before the liquid culture, something I may need to do.

I also just tested a new batch of glycerol and got the same results. I was fairly hopeful that that was the problem, but the new glycerol did not help. I really can't think of why the cells would grow on the plate but not in culture. And why it just started happening all of a sudden. (I grew liquid cultures from glycerol back in July, then the next time in September it failed to work).

Any more ideas?


I would find it pretty weird that they would grow on solid medium, but not in liquid.
Perhaps you did something wrong when preparing the liquid medium?
Are you sure the liquid medium is 100% the same as the plate medium (besides the agar then)?

Comparing the new transfomation with the older stock, does indeed make you believe that the stock is not good anymore, however since the stock does grow on plates... something is not right.
You do aerate them enough (I guess you do since the new ones grow in the liquid)

I do advise you to try what I said: plate them out (first time) and then replate them for a second time before putting them in a broth.

Edited by pito, 19 November 2012 - 11:11 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





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